Download l Saccharomyces cerevisiae as a Genetic Model Organism

Genetic Techniques for Biological Research
Corinne A. Michels
Copyright q 2002 John Wiley & Sons, Ltd
ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)
l
Saccharomyces cerevisiae as a Genetic
Model Organism
OVERVIEW
Baker’slbrewer’syeast, Saccharomyces cerevisiae, is a moleculargeneticmodel
organism. It is a eukaryote with a nucleus and membrane-bound organelleslike
mitochondria, peroxisomes, endoplasmic reticulum, and a Golgi complex. As such,
complex processes like chromosome replication, transcription and translation, cell
division,secretion, membrane trafficking,subcellular compartment structure and
function, energy metabolism, cytoskeletal structure and mechanics, and intracellular
signaling that are carried out byall eukaryotes can beexploredin detail in an
organism with a well-developed and simple-to-use genetic system. Saccharomyces is
easy to culture and obtain in quantity, thus making it amenable to biochemical
analysis. Gene manipulation techniques for Saccharomyces are extremely powerful.
The major disadvantage of working with Saccharomyces is cell size, which makes
cytologicalanalysisdifficult.Nevertheless,
continued developmentof new microscopic techniques and analytical tools has improved the situation greatly. It is likely
that the function ofeachof
Saccharomyces’ 6000+ geneswill soon be known
making Saccharomyces a tool for invivo testing of the function of genes derived
from other organisms with less-well-developed genetic systems. Detailed protocols
for many of the techniques described in Chapter 1 can be found in Section 13 of
Current Protocols in Molecular Biology (Ausubel et al., 2001). Other excellent guides
to yeast genetic methods are The Guide to Yeast Genetics and Molecular Biology
(Guthrie & Fink, 1991), Methods in Microbiology. Vol. 26: Yeast Gene Analysis
(Brown & Tuite, 1998), and Methods in Yeast Genetics (Burke et al., 2000).
CULTURE CONDITIONS
Saccharomyces can be grown in defined media, either liquid or solid, that provide
the energy and nutrients required for growth and proliferation. In a liquid medium,
inwhich the components are dissolvedin water, the individualcells are in suspension. Agar is added to a liquid medium to make solid media. Individual cells
placed on the surface of a solid medium grow and divide many times using the
nutrients that diffuse to them from the surrounding medium.They form welldefined
colonies
that are clones containing billions
of
genetically
identical
individualcells.Dividingcells
are said to bein the logarithmicphase of growth
because the number of cells is doubling at a rate that is dependent on the nutritional quality of the medium.When
one or more essential nutrients become
limiting, growth and division will slow or even stop and the cells are said to be in
stationary phase and the culture isreferred
to as a saturated culture. This
4
GENETIC
TECHNIQUES
FOR BIOLOGICAL
RESEARCH
terminology is most often used to describe a liquid culture, but cells in colonies also
go through similar phases.
Both rich and synthetic minimal media are used to culture Saccharomyces. Rich
medium, called YEP or YP, is made from commercially available yeast extract and
peptone (a complex protein digestion product). It contains all essential nutrients
including
ammonia
(a
rich nitrogen
source),
phosphate,
sulfate,
sodium,
magnesium, calcium, copper, iron, etc. and certain othercompoundsthat
all
Saccharomyces strains are unable to synthesize. In addition, rich medium provides
many macromolecular precursors such as amino acids and nucleotides that wildtype Saccharomyces strains are able to synthesize if necessary. A sugar or other
carbon energy source must be added, such as glucose (dextrose), sucrose, lactic acid,
or others depending on the genotype of the strain and its ability to utilize various
carbon sources. Glucose is the richest and most readily available carbon source and
a rich medium containing glucose is referred to as YEPD or YPD. Because of the
abundant nutrient supply, cells divide rapidly on a rich medium with a division time
of about 90 minutes and easily visible colonies are formed in about 2 days.
Synthetic minimal medium, referred to as SM, is made from commercially available yeast nitrogen base plus a carbon source, usually glucose unless specified. It
provides the essential nutrients listed above but lacks the amino acids, nucleotides,
and other precursors that are in a rich medium. Thus, a strain must be able to
synthesize these in order to grow and divide on SM medium. Growth is significantly
slower on SM medium, with a doubling time of about 4 hours. Saccharomyces can
be grown on a completely chemically defined medium made from about two dozen
organic and inorganic compounds, but for most research this is not necessary. A
strain capable of growing in a defined minimal medium is called a prototrope.
Ideally this minimal medium contains only a carbon source plus inorganic salts, but
it is usual for wild-type microorganisms to require supplements, such as a vitamin,
to this ideal minimal medium. Despite this, the wild-type genotype is generally
considered to be aprototrope.Mutantstrains
unable to synthesize an essential
nutrient are an auxotrope for that particular nutrient.
The following points are very important for the geneticist to note and understand.
If a strain is unable to synthesize a particular essential nutrient, then that nutrient
will have to be added to the synthetic minimal media to allow the strain to grow on
an SM medium. For example, a strain containing amutation in an ADE gene
encoding an enzyme for the biosynthesis of adenine is unable to synthesize adenine
and must have adenine added to the synthetic minimal medium to allow it to grow.
This mutant strain is an adenine auxotrope. Thus, an ade2 mutant strain requires
adenine in the growth medium. Incontrast, if astrain is unable to utilize a
particular carbon source, for example sucrose, then the strain will not be able to
grow on media that provide that carbon source as the sole carbon source. A strain
that contains a mutation in a SUC gene is unable to utilize sucrose because it does
not synthesize functional invertase, the enzyme required to hydrolyze sucrose to
glucose and fructose. Thus, a suc2 mutant strain will not grow if sucrose is the only
carbon source provided and some other carbon source, such as glucose, must be
available. In summary, strains carrying mutations in anabolic pathways require the
product of the pathway for growth while strains carrying mutations in catabolic
pathways cannot grow if the substrate of the pathway is provided.
SACCHAROMYCES
CEREVISIAE
AS A GENETIC MODEL ORGANISM
5
THE MITOTIC LIFE CYCLE
Saccharomyces is a budding yeast, that is, the ovoid (or egg-shaped) mother cell
produces a small protrusion or bud on its surface that grows insize during the
course of interphase of the cell cycle into what will become the daughter cell. After
the S phase is complete and the DNA has been replicated, the nucleus localizes to
the neck region between the mother and the bud, divides into two nuclei, and one
nucleus enters the bud while the other remains in the mother (karyokinesis).
Following karyokinesis the cytoplasms of the mother and daughter cells divide with
the formation of separate plasma membranes and cellwalls (cytokinesis), and
eventually the daughter cell grows to the sizeof the mother. Both cells are then
capable of dividing again. This is outlined in Figure 1.1. A more in-depth description of the cytological changes that occur during mitosis is presented in Chapter 3.
Both haploid and diploid Saccharomyces cell types can divide by mitotic division.
Many eukaryotic organisms favor either the haploid (lower plants, slime molds,
many fungi) or diploid (animals, higher plants) portion of the life cycle and proceed
through the alternate stage very rapidly. For Saccharomyces the existence of stable
haploid and diploid cell types means that the researcher can culture large numbers
of genetically identical individuals (clones) and usethem
for analysis of the
phenotype via cytological or biochemical analysis. Other than dealing with different
numbers of chromosomes, mitosis of diploid and haploid strains is essentially the
same at the levelof
the chromosome. There are some cytological differences
between haploid and diploid cells during mitosis, particularly in bud-site selection,
that are discussed in Chapter 3. These do not affect the genetic analysis of other
traits.
MATING TYPE, MATING, AND THE SEXUAL LIFE CYCLE
In nature most strains of Saccharomyces are diploid and carry the functional allele
of the H 0 gene, homothalic diploids. Laboratory research strains carry mutant ho
and can be grown as stable haploid cells. Haploids occur in two mating types, the a
mating type and the Q mating type, and these differ from one another at a single
locus called the MAT locus. The two alleles of this locus are referred to as MATa
and MATQ. Stable a or Q strains divide mitotically to produce genetically identical
clonesofcells. The existence of a stable haploid stage in the lifecycleof
SLECcharomyces is attractive to the geneticist because strains carrying recessive mutations
can be isolated and identifiedin the haploid cell type and it is not necessary to
inbreed mutagenized cells to obtain a homozygous mutant diploid.
MATa strains mate with MAT& strains by a complex process of cytoplasmic and
nuclear fusion that results in a diploid cell (described inChapter 3). This diploid cell
is also stable and divides by mitosis to produce a genetically identical diploid clone.
The existence of the stable diploid cell type is also extremely useful for the geneticist.
It allows one to determine if a mutant allele is dominant or recessive and it provides
a simple means for carrying out a complementation test. Complementation analysis
is described in Chapter 5 and is used to determine if different mutations map to the
same or different genes.
6
GENETIC TECHNIQUES FOR BIOLOGICAL RESEARCH
@e J
Conjugation
+
@
@a
@
Sporulation
Germmatlon
Germination
’
Figure 1.1 Life cycle of Saccharomyces cerevisiae. Diploid ( a / a )and haploid, a- or a-mating
type, Saccharomyces cerevisiae can reproduce by mitosis to form clones of genetically
identical cells. Haploid cells of opposite mating type can fuse with one another to form an a/
N diploid. When subjected to nutrient starvation conditions,
diploid cells undergo meiosis
producing four haploid meiotic products called ascospores all contained in a single structure
called an ascus. If restored to nutrient sufficient conditions, each of these four ascospores will
germinate and reproduce as haploid cells as follows: two a-mating type cells and two amating type cells. From The Cell Cycle: An Introduction by Andrew Murray and Tim Hunt,
copyright 0 1993 by Oxford University Press, Inc. Used by permission of Oxford University
Press, Inc.
In starvation conditions, the ala diploid undergoes meiotic division and produces
four haploid cells that mature into ascospores. The four haploid products of a single
diploid cell are contained in a sack called an ascus that is designed for survival
under difficult conditions. Using a microdissection device mounted on a microscope
stage, one can separate the individual haploid ascospores and germinate them in
nonstarvation media. Thesewilldivide
to produce geneticallyidentical haploid
clones. This process is shown in Figure 1.l. The simplicity by which the reseacher
can manipulate thesexual life cycle of Saccharomyces is a tremendous advantage for
genetic analysis.
SACCHAROMYCES
CEREVISZAE
AS A GENETIC
MODEL
ORGANISM
7
SACCHAROMYCES GENOME AND NOMENCLATURE
GENOME SEQUENCE
Saccharomyces cerevisiae hasa haploid chromosome number of16. The entire
Saccharomyces genome of strain S288C is sequenced and available on the Saccharomyces Genome
Database
(called SGD)at
http://genome-www.stanford.edu/
Saccharomyces. The site has a variety of tools for sequence analysis thatare
particularly useful for the Saccharomyces researcher, including gene and restriction
maps of the chromosomes. The site is interconnected with genome databases for
other genetic model organisms and sites for protein analysis. There are literature
guides for the known Saccharomyces genes, announcements of interest to the yeast
research community, and contact information for yeast researchers. SGD iswell
worth a visit.
The Saccharomyces genome contains more than 13 million basepairs (13 Mbp)
including the rDNA and more than 6000 open reading frames (ORFs). Each ORF is
named to indicate the chromosome number (A for chromosome Z to P for chromosome XVZ), whether the geneis found on the right (R) or left (L) arm of the
chromosome (that is, to the right or left of the centromere), and the ORF number.
All ORFs are numbered on each chromosome arm starting at the centromere and
goingin the direction of the telomere regardless of which strand is the coding
strand. Finally, the direction of transcription is indicated by a W (for Watson, the
upper strand) or C (for Crick, the lower strand) depending on which strand is the
coding strand. Thus, ORF YBR288C is found on the right arm of chromosome ZZ.
It is the 288th ORF from the centromere, and the lower strand is the coding strand;
that is, it is transcribed from right to left which for the right arm means towards the
centromere.
GENETIC NOMENCLATURE
Saccharomyces gene names consist of three letters anda number (usually 1-3
digits). The letters chosen are most often based on the phenotype or function of the
gene. Note that the number follows immediately after the letters with no space. For
example, a gene encoding one of the enzymes of histidine biosynthesis is referred to
as HZS3. As in all organisms, gene names are italicized. Often, several mutant alleles
of a particular gene have been identified. These can be distinguished by placing a
suffix after the gene name; frequently a hyphen followed by the allele number is
used, all with no spaces.
In Saccharomyces, dominant alleles of a gene are capitalized and recessive alleles
are in lower case. For example, mutant allele #52 is a recessive mutation of URA3
and is written ura3-52. Mutant strains resistant to the toxic effects of the arginine
analogue canavanine carry dominantalterations in CAN1 encoding the arginine
permease and are written CANl-R. It is important that you do not confuse the
concept of a wild-type allele versusa mutant allele with capital letters (for dominant
alleles) versus lower case letters (for recessive alleles). There are many examples of
mutant alleles that are dominant. The interpretation of dominant versus recessive
will be discussed in Chapter 4 in more detail.
8
GENETIC
TECHNIQUES
BIOLOGICAL
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RESEARCH
PHENOTYPE NOMENCLATURE
Descriptive words or abbreviations derived from the gene name can be used when
discussing the phenotype of a strain. For example, a strain carrying a lys2 mutant
allele (genotype Zys2) will not grow in the absence of added lysine. This phenotype
can be referred to as lysine minus, lysine-, or lys-. Note that the letters are not
italicized andthat no gene number is given.Lysine synthesis requires several
enzymatic steps and therefore mutations in any of several genes encoding these
enzymes can cause a lysine minus phenotype. When observing the phenotype of a
strain one has no information as to genotype. Therefore it is inappropriate to use
the gene number. Genotype can only be determined by doing appropriate crosses to
known genetic tester strains.
STRAIN NOMENCLATURE
In journal articles on Saccharomyces itwillbe noted that researchers name their
strains in a wide variety of ways. There are certain standard strains, like S288C,
W303, or YPHSOO, that are commonly used in research laboratories and these will
be referenced in the Materials and Methods section of an article. If the authors have
done some genetic manipulations with these strains, then they will rename the strain
often using their initials. For example, strain YPHSOO was constructed by Phil
Hieter and coworkers, and the letters stand for Yeast Phil Hieter. The article will
statethat the new strain is a derivative of the original strainanda
literature
reference to the original strain will be given. Often a strain list is presented with the
relevant genotype of the strains used in the study along with information on the
derivation of the strain. The genotype will indicate all of the genes that are mutant.
If a gene is not listed it is assumed to be the wild-type allele found in the strain from
which the mutant was derived, such as S288C. While all of these strains are highly
similar at the sequence level they are not identical. Strain differences may be very
few but could potentially besignificant for the particular research project being
described. Geneticists pay very careful attention to strain backgrounds and do their
best to keep them constant.
PROTEIN NOMENCLATURE
The protein product of a Saccharomyces gene can be named based on the gene name
or the function, if it is known. For example, GALZ encodes galactokinase, the first
enzyme in the catabolism of the sugar galactose. The product of the GALZ gene is
referred to as galactokinase, Gall protein, or Gallp. Note that only the first letter is
capitalized and that the protein name is not italicized.
GENETIC CROSSES AND LINKAGE ANALYSIS
Saccharomyces diploids undergo meiosis when placed in starvation conditions and
form four haploid ascospores, or just spores for short, all contained in a single sack
called an ascus. These four spores are referred to as a tetrad sinceeach spore
SACCHAROMYCES
CEREVISIAE
AS A GENETIC
MODEL
ORGANISM
9
containsonechromatid
from each of the 16 tetrads of chromatids found in
prophase I of meiosis.
To fully understand the crosses outlined below, it would be helpful to first review
the process of meiosis including chromatid segregation patterns and independent
assortment, i.e. basic Mendelian genetics. The genetic cross is a powerful tool. In the
initial stages of a genetic analysis the researcher must know whether a single
mutation is producing the mutant phenotype under investigation. A simple genetic
cross can demonstrate this. Crosses are used to construct heterozygous diploids to
determine whether amutation
is dominant or recessive and can beused
to
demonstrate linkage. As can be seen from the literature, linkage analysis is used in a
variety of ways and not simply to map genes on a chromosome.
SINGLE GENE CROSS
If two haploid strains carrying different alleles of the same gene are mated and the
resulting diploid sporulated, the two alleles will segregate to different spores. The
resulting four-spored tetrad will consist of two spores containing one allele of the
gene and two spores containing the second allele ofthe gene. This is shown below in
Cross 1 in which a strain containing a recessive mutation, genl-62, is crossed to a
strain carrying the wild-type dominant allele GENl.
Cross 1:
GENl
(wild-type)
Diploid:
GENI
genl-62
(wild-type)
2:2 Single
Spore
A
B
C
D
x
genl-62 (genotypes of parental strains)
(mutant) (phenotype of parental strains)
(genotype of diploid)
(phenotype of diploid)
gene segregation:
Genotype
Phenotype
GENl
GENI
genl-62
genl-62
wild-type
wild-type
mutant
mutant
Since genl-62 is a single alteration in the GENl gene, the only possible tetrad that
can result from this cross is one containing two GENl spores and two genl-62
spores. If a thousand tetrads were dissected all would be 2 wild-type: 2 mutants
because only a single mutant gene is segregating in this cross. Thus, the 2 : 2
segregation pattern is consistent with the fact that a single genetic difference exists
between the parent strains in the cross.
But what if one does not know whether a mutant strain contains a single mutant
alteration? Perhaps two or more mutations in different genes are needed to produce
the mutant phenotype observed. Perhaps the mutantstrain
has a complex
phenotype and exhibits several abnormalities. Are all these abnormal phenotypes
associated with a single gene mutation or are there several mutations in the strain
10
GENETIC
TECHNIQUES
BIOLOGICAL
FOR
RESEARCH
each causing a specific phenotype? These questions can be answered by crossing the
mutant strain to a wild-type strain (one that has not been exposed to mutagenesis
and selection). If tetrad analysis of many tetrads derived from this heterozygous
diploid gives only two mutant spores and two wild-type spores, then this is strong
evidence that one is working with a mutant strain carrying a mutation in a single
gene. If all the mutant phenotypes are exhibited by all the mutant spores, then one
can conclude that the single gene mutation has several phenotypic effects, i.e. it is
pleiotropic. If one finds some other segregation pattern, such as one mutant spore to
three wild-type spores, or if the phenotypes segregate from one another, then one
must consider the possibility that the mutant strain contains mutations in two or
more genes (see Cross 3 below). There are other interpretations of a 1 : 3 ratio, such
as a high rate of gene conversion or aneuploidy, but these are less likely particularly
if the mutations were induced by a mutagen and are not spontaneous.
The ‘single gene cross’ has other uses. If one crosses two mutant strains believed
to contain different mutant alleles of the same gene, such as a deletion and a single
base change, then these alleles should always segregate to different spores. All of the
haploid spores resulting from the heterozygous diploid should contain either one or
the other mutant allele. Given this segregation pattern, only tetrads containing four
mutant and nowild-type will result, except for rare recombinants between the alleles
if recombination is possible. Thus, a 4 : 0 result in all tetrads is strong evidence that
the mutations are in the same gene, or extremely closely linked genes. This is shown
in Cross 2 below.
Cross 2:
genl-33
x genl-62
(mutant)
(mutant)
(phenotype
Diploid:
genl-33
genl-62
(mutant)
2:2 Single
Genotype
Spore
A
B
C
D
(genotypes of parental strains)
of parental strains)
(genotype of diploid)
(phenotype of diploid)
gene segregation:
Phenotype
genl-33
genI -62
genl-33
genI-62
mutant
mutant
mutant
mutant
TWO GENE CROSS
If strains carrying mutations in two different genes are crossed, then the genes will
recombine producing recombinant meiotic products with a wild-type and double
mutant genotype. The frequency of recombination will depend on whether the genes
are linked (map close to one another on the same chromosome) and, if linked, how
tightly they are linked. When the genotypes of the spores in different tetrads from
such a diploid are determined, three classes of tetradsareobtained
as follows.
SACCHAROMYCES
CEREVISIAE
AS A GENETIC MODEL
ORGANISM
11
Parental ditype (PD) tetrads resultwhen no recombination occursbetween the
mutant genes during meiosis of the diploid cells. These will contain four spores, two
ofeach parental genotype.When recombination occurs during meiosisof the
diploid cell, either tetratype (TT) or nonparental ditype (NPD) tetrads are obtained.
A tetratype tetrad contains four spores each with a different genotype, including the
two parental genotypes and the two recombinant genotypes, which are wild-type
and the double mutant. A nonparental ditype tetrad contains two types of spores
neither of which is the parental genotype, i.e. both are recombinant types, including
two wild-type spores and two double mutant spores. This is shown below in Cross
Cross 3:
genlGEN2
(mutant)
(mutant)
Diploid:
genl
- gen2
- (genotype)
GENl
GEN2
(wild-type)
(phenotype)
Parental ditype:
Spore
Genotype
A
B
C
D
GEN2
genl
GEN2
genl
GENl gen2
GENI gen2
Tetratype:
Spore
Genotype
A
B
C
D
GEN2
genl
genl gen2
GENI gen2
GEN2
GENl
Nonparental ditype:
Spore
Genotype
A
B
C
D
genl gen2
genl gen2
GEN2
GENl
GEN2
GENl
X
GENI gen2
(genotypesof parental strains)
(phenotype of parental strains)
Phenotype
mutant
mutant
mutant
mutant
Phenotype
mutant
mutant
mutant
wild-type
Phenotype
mutant
mutant
wild-type
wild-type
The frequency of each type of tetrad will depend on the frequency of recombination. If the two mutant genes are completely unlinked, that is 50% recombination,
then the frequency of PD : TT : NPD tetrads will be 1 :4 : 1. If there is any linkage,
that is the frequency of recombination is less than 50%, then the relative number of
PD tetrads willincrease to greater than the expected 1/6 of the total number of
tetrads analyzed and the number of PD tetrads willexceed the number of NPD
GENETIC
TECHNIQUES
BIOLOGICAL
12
FOR
RESEARCH
tetrads. Ultimately, for crosses between two alleles, 100% of the tetrads will be PD,
as is shown in Cross 2.
It is important to note that the phenotype of the double mutant may be unique.
This occurs most often when the two genes encode functions involved in the same
process. If mutant strains containing alterationsin unrelated gene functions, such as
ade2 and suc2, are crossed, then the doublemutant is expected to exhibit both
phenotypes, adenine requiring and unable to utilize sucrose. If mutantstrains
containing alterations in two related gene functions are constructed, such as MCM2
and MCM7 encoding different components of the origin recognition complex
(ORC), then the doublemutant
could exhibit an unexpected phenotype. For
example, the double mutant combination could be lethal even though each single
mutantstrain
is viable. Often mutant genes are crossed for the purpose of
determining the doublemutant phenotype. Aswillbe
discussedin detail in the
chapters on epistasis, suppression, and enhancement, a great deal of insight into the
function and relationship between gene products can be obtained from observing
the phenotype of the double mutant.
CLASSES OF SACCHAROMYCES CLONING PLASMID
VECTORS
Saccharomyces plasmids were developed from Escherichia coli plasmid vectors. The
basic E. coli vector is small [2-4 kilobasepairs (kbp) of DNA] and includes genes
needed for plasmid replication, an origin of replication (ORI) derived from an E.
coli plasmid, and a selectable marker gene such as AMP' (for ampicillin resistance)
to be used to identify E. coli transformants containing the plasmid. The E. coli OR1
allows the plasmid to replicate independent of the E. coli chromosome as an extrachromosomal element or plasmid. As such it is easy to purify in large amounts.
Additionally, one or more restriction sites will be present for cloning foreign DNA
sequences.
E. coli plasmids are the foundation for the construction of the Saccharomyces
yeast cloning vectors. Saccharomyces sequences were added to the E. coli vectors to
create what are referred to as E. colilyeast shuttle vectors, meaning that these
plasmid vectors are able to establish themselves in either organism. First, a marker
gene capable of being selected in a yeast host strain was included in order to be able
to select yeast transformants. Good antifungal agents, comparable to the ampicillin
and tetracycline used in E. coli, were not initially available. Therefore, nutritional
genes encoding enzymesin biosynthetic pathways were the first to beused as
selectable marker genes in Saccharomyces. More recently antifungal agents like
kanamycin (also called G418 or neomycin) and hygromycin have come into use.
URA3, LEU2, T R P I , and HIS3 are the genes most commonly used as selectable
marker genes for Saccharomyces transformation. The Saccharomyces strains used as
hosts for plasmid vectors carrying these nutritionalmarker genes must contain
recessive mutant alleles of these genes in order to be an appropriate host. Suitable
mutant alleles of URA3, LEU2, TRPI, HIS3 and other genes are available. Strains
like YPHSOO have been specially constructed to carry several of these mutant genes.
It is important to keep in mind that transformation is rare, about one in 1000 cells
SACCHAROMYCES
CEREVISIAE
AS A GENETIC
MODEL
ORGANISM
13
or less, and not so different from the rate of mutation. To facilitate selection of
transformants as opposed to the back mutations to wild-type, the mutant alleles of
these genes do not revert at any appreciable rate because they are deletions, multiple
point mutations, or transposon insertion mutations.
A typical Saccharomyces transformation is carried out as follows. An appropriate
hosthector pair is selected. For example, a host strain carrying the ura3-52 allele is
unable to grow ona minimal medium that lacks uracil becauseit is unable to
synthesize uracil, whichis essential for various cellular processes including RNA
synthesis. If a plasmid carrying the wild-type dominant URAS gene is introduced
into this host strain by transformation, then the transformant will be able to grow
on a minimal medium lacking uracil. The plasmid DNA is transformed into the host
cells by any one of a number of methods including chemical treatments, electroporation, or pellet guns. The DNA treated cells are plated ona solid synthetic
medium lacking uracil. Only those individuals that have acquired a stable copy of
(IRA3 by transformation with the plasmid vector will be able to form colonies. Of
course this must be confirmed by appropriate tests.
Thefate of the plasmid after entering a Saccharomyces cell depends on the
particular Saccharomyces sequences it contains. If a Saccharomyces origin of
replication is included, then the plasmid will replicate as an extrachromosomal
element. Its copy number, the average number of plasmids per cell, is determined in
part by the class of Saccharomyces OR1 and whether or nota Saccharomyces
centromere is also included in the plasmid. If the plasmid vector lacks a Saccharomyces replication origin, then the plasmid must integrate at a chromosomal
site(usually by homologous recombination between vector sequences and the
chromosome) to produce a stable transformant. If the plasmid vector integrates,
then it will replicate as part of the chromosome.
YIP PLASMID
A YIP plasmid consists of the basic E. coli vector described above plus a Saccharomyces selectable marker gene, but does notcontaina
Saccharomyces origin of
replication. Therefore, YIP plasmids must integrate into a chromosome in order to
be replicated at each cell division. If integration does not occur, the transforming
DNA will be lost due to degradation or dilution by cell division.
Integration occurs by means of a single crossover (recombination) event between
the plasmid DNA and the chromosome. This is illustrated below in Figure 1.2. The
crossover occurs only between homologous DNA sequences and is carried out by
the generalized recombination enzymes. After the integration event, the plasmid
sequences arepart of the chromosome, are replicated when the chromosome is
replicated, andare passed intoboth the mother and daughter cells during cell
division, as are all the other sequencesof the chromosome. Figure 1.2 shows a
recombination event occurring between a yeast sequence carried by this vector and a
homologous chromosomal sequence. This recombination event might also have
occurred between the URAS sequence on the plasmid and the mutant ura3-52 gene
in the host since sequences are still present at this site. To prevent this, one could use
ura3 deletion mutation.
14
GENETIC TECHNIQUES FOR BIOLOGICAL RESEARCH
,,/
-.
,....+_._._..
uRA3
'-'-'-.-..,
'%,
Figure 1.2 Targeted integration
The integration of a YIP plasmid can be targeted to a specific chromosomal site
(shown in Figure 1.2). Integration requires recombination between homologous
sequences on the plasmid and chromosome. For supercoiled plasmid DNA, this
recombination occurs in about onein 10 000-100000 transformed cells. But, if prior
to transformation the plasmid is digested with a restriction enzyme that cuts ata site
within the homologous sequence creating a highly recombinogenic double-strand
break, then the frequency of recombination will increase about 1000-fold. Therefore, as is shown in Figure 1.2, if a particular YIP plasmid carrying two Succharomyces sequences, for example URA3 and LEU2, is digested at a site in the LEU2
gene and transformed into a uru3 leu2 host strain, then it will integrate 1000 times
more often at the leu2 locus than at the u r d locus. If transformant strains are
crossed to another strain of opposite mating type with the uru3 leu2 genotype and
sporulated, then all of the tetrads will be PD with two uracil- leucine- spores and
two uracil+ leucine+ spores. That is, the URA3 LEU2 alleles will segregate together
because they are both linked to the site of plasmid integration.
YRp PLASMID
YRp plasmids are constructed from the
basic YIP vector by the addition of a
Saccharomyces origin of replication derived from a chromosomal sequences. These
yeast OR1 sequences are commonly called ARS sequences for autonomously replicating sequence. Chromosomal replication initiates at these sites and, on average,
they are found every 40 kilobasepairs of DNA in Saccharomyces. YRp plasmids can
be integrated but normally they are not and are able to
replicate as independent
extrachromosomal plasmids. Depending on the particular ARS element, they are
present in 5-10 copies per cell on average.
YRp plasmids are unstable because there is no mechanism to move the plasmid
copies into the bud (like a spindle) and they often get left behind in the mother
nucleus. Because of this, the growth ofcells transformed with YRp plasmids in
nonselective media (that is, media containingthenutrient
synthesized by the
SACCHAROMYCES CEREVZSZAE AS
GENETIC
A
MODEL
ORGANISM
15
selection marker carried on the plasmid) leads to the spontaneous lossof the
pksmid. To ensure that the plasmid is maintained by most of the cells in a culture,
transformants must be grown in a selection medium lacking the nutrient at all times
so that only cells inheriting the plasmid will be capable of growing and dividing.
YEp PLASMID
A YEp plasmid containsan
origin of replication derived from the naturally
occurring Saccharomyces plasmid called the 2 p circle in the basic YIP vector. The
Saccharomyces 2p circle is a very abundant plasmid found in many natural strains
and most laboratory strains. The 2p circle OR1 is a very active origin and YEp
plasmids normally replicate as high-copy independent extrachromosomal plasmids
present in25-50 copies per cell on average. YEp plasmids are also unstable and
transformants must be grown under selection to maintain the plasmid.
YCp PLASMID
A YCp plasmid contains a centromere sequence, CEN, derived from one of the 16
Saccharomyces chromosomes added to a YRp vector. These plasmids are treated
like mini chromosomes by the dividing Saccharomyces cell. YCp plasmids attach to
spindle fibers during division and are very efficiently transmitted to both mother
and daughter cells in mitosis and meiosis, although not as efficiently as the normal
chromosomes. Therefore, YCp plasmids are very stable plasmids that replicate and
segregate along with the remainder of the chromosomes. As a result, YCp plasmids
are low-copy independent extrachromosomal plasmids present in 1-2 copies per cell
on average and are lost from transformant cells at a verylow rate evenin the
absence of nutritional selection.
YAC PLASMID
YAC vectors are designed to carry large chromosomal fragments of DNA and have
been very useful in cloning fragments for various genome sequencing projects and
for positional cloning studies. YAC stands for yeast artificial chromosome. The
major difference between YAC vectors and YCp vectors is the inclusion of two
copies of a sequence derived from Saccharomyces telomere DNA consisting of many
repeats of the short nucleotide sequence 5'C2-3A(CA)I-3 on one strand with the
complementary GT-rich sequence repeated on the other strand. In the circular YAC
vector plasmid, the two copies are separated by a stuffer fragment that is cut out
using restriction enzymes prior to transformationinto the host Saccharomyces
strain. Once in the host cell, endogenous telomerase enzyme will elaborate a full
telomere at each end of the linearized YAC vector DNA. Natural Saccharomyces
chromosomes range from 230 to 1700 kbpbut
these have several origins of
replications each. Inserts up to 1400 kbp can be accommodated in certain YAC
vectors.
16
GENETIC
TECHNIQUES
FOR BIOLOGICAL
RESEARCH
LIBRARIES
Saccharomyces plasmid libraries can be constructed from any of these types of
vector. The choice depends on whether a stable or unstable Saccharomyces transformant is desired and whether one or many copies per cell are needed.
GENE DISRUPTIONlDELETION IN SACCHAROMYCES
(ONE-STEP GENE REPLACEMENT)
Gene disruption is a method by which a DNA fragment is used to replace a genome
sequence with a selectable marker gene, such as HIS3 or kanavanine resistance. In
so doing, a deletion is created. The process occurs by homologous recombination
and uses the enzymes of the homologous recombination pathway, such as Rad52p.
Theends of the exchange fragment must be long enough and have sufficient
homology to the chromosomal site so that homologous recombination can occur.
Moreover, the sizeof the region to be deleted can be quite large but must be
contained in a single chromosome.
One-step gene replacement is a relatively efficient process. Free DNA ends are
very ‘recombinogenic’ in yeast. This means that free 3‘ and 5‘ ends of doublestranded DNA fragments in vivo search out homologous sequences in the chromosomes with very high efficiency. When a homologous sequence is found, the free
ends invade the chromosomal sequence and this leads to a crossover event at a site
near the free end. If this happens at both ends of a DNA fragment, then the
fragment replaces the genomic copy. This is shown in Figure 1.3 for a fictitious
gene, YGII (Your Gene of Interest).
Recombinant DNA methods can used to construct the disruption fragment. This
method was used prior to the development of polymerase chain reaction (PCR)based methods (see below) and is often seen in the literature. Disruption constructs
for many genes are available from researchers in the yeast community and are
provided upon request. To make a disruption construct, one starts with the cloned
genomic fragment (contained in an E. coli plasmid vector). Restriction digestion or
other related methods can beused to cut out the internal sequences and replace
them with the selectable marker gene. This is shown in Figure 1.4.
More recently, PCR-based methods have been used for the construction of the
disruption fragment. In yeast, only about 40 bp of sequence are needed at each end
of the disruption fragment in order for the crossover events to occur properly, but
the sequence must be identical to the genomic target sequence. The PCR primers are
used to amplify the selection gene and place a target site sequence at either end.
Each primer consists of 40 bp of target site sequence at the 5‘ end followed by a
short sequence homologous to the selection gene. The complete selection marker
gene must be amplified, including the promoter and ORF. Longtine et al. (1998)
describe plasmid constructs designed specifically to provide selection marker templates for PCR amplification. The kanMX6 resistance gene and the Saccharomyces
pombe h i s 9 genefused to appropriate S. cerevisiae promoterandterminator
sequences as well as the S. cerevisiae TRPl gene are available in this series. The
kanMX6 and his5+ genes are particularly useful because they lack homology to S.
SACCHAROMYCES CEREVZSZAE AS
A
___-_ I
Crossover
I
17
GENETIC MODEL
ORGANISM
I
YGIl
----_ -Chromosome
Crossover
Disruption fiagment
Transformation of his3 host strain
with disruption fiagment
Select for His+ transformants
Chromosome
-------_-__
Figure 1.3 One-step gene disruption of YGIl
Cloned genomic fragment
Restriction
Restriction
i.
\
i
HZS3 gene fiagment
i
Disruption hgment
Figure 1.4 Construction of a disruption fragment using available restriction sites
cerevisiae genomic sequences and thus preclude the possibility of recombination at
sites in the genome other than at the intended disruptionsite. When deleting a gene,
it is best to remove sequences starting in the promoter and extending into the
ORF or past the stop codon. This
ensures that the gene has been functionally
knocked out. If the transcription and translation startsites are not removed and the
deletion is internal to the ORF, it is conceivable that some gene function could be
retained.
Whetherthetraditional
or thePCR-basedmethod is used for one-step gene
disruption, it is important to confirm that the event has occurred correctly. This can
be done by Southern analysis or by PCR of genomic DNA using onesprimer that
anneals to sequences outside the deleted region and one primer that anneals to
internal sequences in the selection marker gene.
GAP REPAIR
This is a method frequently used to recover a specific sequence from the chromosome onto an episomal plasmid. Gap repair utilizes the host cell’s recombination/
repair and DNA replication machinery to fill an artificially created deletion in a
GENETIC TECHNIQUES FOR BIOLOGICAL RESEARCH
Figure 1.5 Gap repair
homologous sequence carried on the plasmid. Its most common use is for cloning
different alleles of a cloned gene.
One startswith the DNA fragment of interest cloned into a plasmid vector that is
maintained as an extrachromosomal element, such asYRp
orYEP.
Using
restriction endonucleases that cut sitesin the insert fragment but not in vector
sequences, one creates a deletion internal to the yeast DNA fragment. It is essential
to leave at least 50 bp of insert fragment at either end to provide homology to the
chromosomal site as asubstratefor
recombination. This linearized and gapped
fragment is then transformed into the host cell and transformants are selected using
the marker gene carried by the plasmid vector. For the example shown in Figure
1.5, the host strain is ura3 and repair of the gap is essential if the cell is to maintain
the plasmid and to be able to grow on a selection medium lacking uracil. The
gapped region is filled by a gene-conversion-like event between the gapped plasmid
and the homologous chromosomal site. The arrows in Figure 1.5 indicate the endpoints of the gap and the positions where the exchange events will initiate. The free
ends of the gapped fragment invade the homologous chromosomal sequence, DNA
replication of the gapped region occurs from these ends using the chromosomal
sequence as template, and the gap is filled.
Gap repair is used to recover different alleles of the cloned sequence from the
chromosome. For example, one has cloned the wild-type allele of a gene and wants
to clone the available mutant alleles. Another use of gap repair is in fine structure
mapping of recessive mutant alleles. If a mutation maps outside the gapped region,
then filling in the gapped region of the wild-type allele carried on the plasmid with
the chromosomal sequence will result in the restoration of the wild-type allele on the
plasmid copy of the gene and stable transformants with the wild-type phenotype of
the gene of interest will result. If the mutation maps within the gap, then only stable
transformants with the mutant phenotype will be obtained.
REPORTER AND OTHER TYPES OF FUSION GENE
A reporter gene is used to follow gene expression in vivo. It is a fusion between all or
part of a gene of interest with another gene whose product is easy to detect or
measure qualitatively and/or quantitatively. Most often, the researcher will choose
to use a reporter gene if the product of the gene of interest is difficult to assay or
detect. Thus, the reporter gene product acts as a surrogate.
AS A GENETIC MODEL
ORGANISM
SACCHAROMYCES
CEREVISIAE
~~~~
YGIl gene
YGIl promoter
YGIl (OW)
19
1
Reporter gene (OW only)
Figure 1.6 Reporter gene fusion constructions
A fusion gene between the gene of interest and the reporter gene can include only
the upstream promoter of the gene of interest or part or all of its ORF. If a coding
region is included, then the sequence at the fusion junction must maintain the
correct reading frame so that a single ORF is produced that encodes a fusion of the
two proteins. This is shown in Figure 1.6. Fusion gene constructions are often
carried on plasmid vectors but they can also be integrated into chromosomes,
depending on the needs of the experiment.
There are several commonly used reporter genes including lacZ (encoding pgalactosidase from E. coli), CAT (encoding chloramphenicol acetyltransferase, the
bacterial protein providing chloramphenicol resistance), luciferase gene (encoding
the phosphorescent protein from firefly), and GFP [encoding green fluorescent
protein (GFP) from a jellyfish]. To be useful, the host organism must not encode a
protein with the same activity, otherwise one could not be sure whether one was
observing the activity of the endogenous protein or the reporter protein. Using a
variety of techniques, these proteins can be measured either in vivo or in vitro. pGalactosidase can be assayed in vitro using cell extracts by measuring the rate of
hydrolysis of an uncolored compound called ONPG to a yellowdye or another
uncolored compound called X-gal to a blue dye. The X-gal reaction is particularly
useful because is can be done on whole cells in tissues or colonies growing in a petri
dish. Cells expressing the @galactosidase reporter will be bright blue. GFP is very
useful for determining the subcellular location of a protein and the type of fusion
used for this analysis is an in-frame fusion between the full-length gene of interest
and the GFP gene (discussed in detail in Chapter 2). Alternately, portions of the
gene of interest can be fused to GFPto localize the portion of the protein of interest
responsible for targeting the protein to a particular subcellular compartment. There
are many other uses of these types of construction.
A variety of E. colilyeast shuttle vectors are available for the construction of
fusion genes. These contain a multiple cloning sequence at the junction site of the
fusion. The DNA sequence to be fused to the vector gene, whether it is a promoter
or an ORF, is typicallyamplified by PCR using primers that place appropriate
restriction sites at the ends of the fragment. The fragment is then cloned into the
multiple cloning site to create the fusion. PCR-based methods are also available for
20
GENETIC
TECHNIQUES
BIOLOGICAL
FOR
RESEARCH
creating fusions at sites in the genome and these are described in Chapter 2
(Longtine et al., 1998).
EXPRESSION VECTORS
Expression vectors are vectors that allow one to construct gene fusions that replace
the native promoter of a gene with another promoter forany of a variety of reasons.
For example, the native promoter might initiate transcription at a very low rate, too
low to allow for purification or detection of the protein product of the gene, or only
under very special conditions. Placing the ORF of the gene of interest under the
control of a high-level constitutive promoter in aYEp vector would increase
expression of the protein hopefully to levels that wouldallow the researcher to
purify and characterize the product.
Several expression vectors are available to the Saccharomyces researcher and can
be obtained from colleagues or from commercial sources. The ADHl promoter is
commonly used for high-level constitutive expression in glucose-grown cells. GALl
and GAL10 are frequently used when regulated expression is desired. The GALl and
GAL10 are induced to veryhighlevelsin
galactose grown cells but expression is
dramatically repressed by growth on glucose.
An expression system developed by Mumberg et al. (1995)allows for the constitutive production of a gene product over a 1000-fold range. One can choose from
the promoters of either CYCl encoding cytochrome-c oxidase isoform 1, ADHl
encoding alcohol dehydrogenase 1, TEF2 encoding translation elongation factor la,
or GPDI encoding glyceraldehyde-3-phosphate dehydrogenase. These are available
in either YEp or YRp vectors, which provides another mechanism for varying the
expression level. Additionally, one can choose from either HIS3, LEU2, URA3, or
TRPl as the selectable marker. If one prefers to be able to regulate the expression of
the gene of interest, Labbe & Thiele (1999) developed a similar vector series but use
the CUPI, CTRI, and CTR3 copper-regulated promoters.
REFERENCES AND FURTHER READING
Ausubel,F.M.,R.Brent,R.E.Kingston,
D.D.Moore,J.G.Seidman,J.A.Smith,
& K.
Struhl, editors (2001) Current Protocols in Molecular Biology. John Wiley & Sons, Ltd.,
New York.
Brown,A.J.P. & M. Tuite (1998) Methods in Microbiology, Vol. 26: Yeast Gene Analysis.
Academic Press, New York.
Burke, D., D. Dawson, & T. Stearns (2000) Methods in Yeast Genetics. Cold Spring Harbor
Press, New York.
Guthrie,C. & G.R.Fink,editors (1991) Guide to Yeast Genetics and Molecular Biology.
Methods in Enzymology, Vol. 194. Academic Press, New
York.
Labbe, S. & D.J. Thiele (1999) Copper ion inducible and repressible promoter systems in
yeast. Methods Enzym. 306: 145-153.
Longtine,M.S., A. McKenzie 111, D.J. Demarini, N.S. Shah, A. Wach, A. Brachat, P.
Philippsen, & J.R. Pringle (1998) Additional modules for versatile and economical PCRbased gene deletion and modification in Saccharonzyces cerevisiae. Yeast 14: 953-961.
SACCHAROMYCES
CEREVISIAE
AS A GENETIC
MODEL
ORGANISM
21
Mumberg, D., R. Muller, & M. Funk (1995) Yeast vectors for the controlled expression of
heterologous proteins in different genetic backgrounds. Gene 156: 119-122.
Murray, A. & T. Hunt (1993) The Cell Cycle. An Introduction. Oxford University Press, New
York.
Walker, G.M. (1998) YeastPhysiology and Biotechnology. John Wiley & Sons,Ltd., New
York.